Rebeccamycin analogs by tryptophan analogs feeding

This application is a continuation of application Ser. No. 08/060,951, filed May 13, 1993, now abandoned, which is a continuation of application Ser. No. 07/648,751, filed Feb. 5, 1991, now abandoned, which is a CIP of application Ser. No. 07/489,430 filed Mar. 6, 1990, now abandoned.

1. Field of the Invention

This invention relates to novel analogs of rebeccamycin which posses antineoplastic properties.

2. Background Art

U.S. Pat. Nos. 4,487,925 and 4,552,842 disclose the anti-tumor agent designated rebeccamycin, and the 5'-methyl and 5',2',3",6"-tetraacetate derivatives thereof, and a process for producing the same agent by cultivating a rebeccamycin-producing strain of Nocardia aerocolonigenes, preferably Nocardia aerocolonigenes ATCC 39243, or a rebeccamycin-producing mutant thereof in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a substantial amount of rebeccamycin is produced. Recently, Nocardia aerocolonigenes, ATCC 39243 was reclassified as Saccharothrix aerocolonigenes, ATCC 39243 (Bush et at, J. Antibiotics, 40:668-678, 1987).

The present invention provides new analogs of the antitumor agent designated rebeccamycin (Formula I). ##STR1##

More specifically, there are provided rebeccamycin analogs of the Formulas II and III below, ##STR2## wherein X 1 and X 2 are independently fluorine or hydrogen, provided that X 1 and X 2 are not simultaneously hydrogen; as well as pharmaceutically acceptable acid addition salts of such analogs.

The compounds of the Formulas II and III are produced by supplementing the cultures of a rebeccamycin producing strain of Saccharothrix aerocolonigenes with the appropriate tryptophan analog.

FIG. 1 shows the IR spectrum for the compound of Formula IV.

FIG. 2 shows the 1 H-NMR for the compound of Formula IV.

FIG. 3 shows the 13 C-NMR for the compound of Formula IV.

FIG. 4 shows the IR spectrum for the compound of Formula V.

FIG. 5 shows the 1 H-NMR for the compound of Formula V.

FIG. 6 shows the 13 C-NMR for the compound of Formula V.

FIG. 7 shows the IR spectrum for the compound of Formula VIII.

FIG. 8 shows the 1 H-NMR for the compound of Formula VIII.

FIG. 9 shows the 13 C-NMR for the compound of Formula VIII.

FIG. 10 shows the mass spectrum of the compound of Formula IV.

FIG. 11 shows the UV spectrum of the compound of Formula IV.

FIG. 12 shows the mass spectrum of the compound of Formula V.

FIG. 13 shows the UV spectrum of the compound of Formula V.

FIG. 14 shows the mass spectrum of the compound of Formula VIII.

FIG. 15 shows the UV spectrum of the compound of Formula VIII.

U.S. Pat. Nos. 4,487,925 and 4,552,842 disclose the production and isolation of the antitumor agent designated rebeccamycin (Formula I) ##STR3##

The above-mentioned rebeccamycin compound is the principal component of the fermentation of the rebeccamycin producing strain of Saccharothrix aerocolonigenes.

It has now been found according to the present invention that the fermentation procedure disclosed in U.S. Pat. Nos. 4,487,925 and 4,552,842 can be carried out in the presence of certain tryptophan analogs to produce new analogs of rebeccamycin having valuable antitumor properties. The rebeccamycin analogs of the present invention have the Formulas II and III below. ##STR4## wherein X 1 and X 2 are fluorine or hydrogen, provided that X 1 and X 2 are not simultaneously hydrogen; as well as pharmaceutically acceptable acid addition salts thereof.

A preferred example of the compounds of the present invention is the compound having the structural formula IV below: ##STR5##

Another preferred example of the compounds of the present invention is the compound having the structural formula V below: ##STR6##

Another preferred example of the compounds of the present invention is the compound having the structural formula VI below: ##STR7##

Still another preferred example of the compounds of the present invention is the compound having the structural formula VII below: ##STR8##

Still another preferred example of the compounds of the present invention is the compound having the structural formula VIII below: ##STR9##

Another preferred example of the compounds of the present invention is the compound having the structural formula IX below: ##STR10##

Another preferred example of the compounds of the present invention is the compound having the structural formula X below: ##STR11##

Yet another preferred example of the compounds of the present invention is the compound having the structural formula XI below: ##STR12##

In the present process a tryptophan analog is added to the rebeccamycin fermentation medium and is incorporated during fermentation into the rebeccamycin structure creating a corresponding rebeccamycin analog. A more extensive description of the process is given below and in the illustrative examples which follow.

Preparation of the Antibiotics

The compounds Formulas IV-XI are produced by cultivating a rebeccamycin producing strain of Saccharothrix aerocglonigenes, with DL-4-fluorotryptophan, DL-5-fluorotryptophan, DL-6-fluorotryptophan, or DL-7-fluorotryptophan. The preferred producing organism is a novel strain of Saccharothrix aerocolonigenes, previously designated as Nocardia aerocolonigenes strain C38,383-RK2 (ATCC 39243) in U.S. Pat. No. 4,487,925. Recently, this strain was reclassified as Saccharothrix aerocolonigenes (Bush et al., J. Antibiotics 40:668-678, 1987) and is designated herein as Saccharothrix aerocolonigenes strain C38,383-RK2 (ATCC 39243). This strain was isolated from a soil sample collected in Panama. A biologically pure culture of strain C38,383-RK2 has been deposited with the American Type Culture Collection, Rockville, Md., and added to their permanent collection of microorganisms as ATCC 39243. This culture, designated as C38,383-RK2, is also maintained as a dormant culture in lyophile tubes and cryogenic vials in the Bristol-Myers Squibb Co. Pharmaceutical Research and Development Division Culture Collection, 5 Research Parkway, Wallingford, Conn. 06492.

The taxonomic studies on strain C38,383-RK2 (ATCC 39243) have been described in detail in U.S. Pat. No. 4,487,925 and in J. Antibiotics 40:668-678, 1987. The strain has been classified as a novel strain of Saccharothrix aerocolonigenes.

It is to be understood that the present invention is not limited to use of the particular preferred strain ATCC 39243 or to organisms fully answering its description. It is especially intended to include other rebeccamycin producing strains or mutants of the described organism which can be produced by conventional means such as x-radiation, ultraviolet radiation, treatment with nitrogen mustard, phage exposure and the like.

In practicing the present process, a rebeccamycin-producing strain of Saccharothrix aerocolonigenes, having the identifying characteristics of strain C38,383-RK2 (ATCC 39243), or a mutant or variant thereof, is cultivated in a conventional aqueous nutrient medium supplemented with the appropriate tryptophan analog. For optimal production of the compound of Formulas VI and X, the medium should be supplemented with DL-4-fluorotryptophan. For optimal production of the compounds of Formulas IV and IX, the medium should be supplemented with DL-5-fluorotryptophan. For optimal production of the compounds of Formula V and VIII, the medium should be supplemented with DL-6-fluorotryptophan. For optimal production of the compounds of Formulas VII and XI, the medium should be supplemented with DL-7-fluorotryptophan. The organism is grown in a nutrient medium containing known nutritional sources for actinomycetes. Thus, the organism is grown in a nutrient medium containing an assimilable carbon source such as sucrose, lactose, glucose, rhamnose, fructose, glycerol or soluble starch. The medium should also contain an assimilable nitrogen source such as fishmeal, peptone, peanut meal, cottonseed meal, corn steep liquor, amino acids or ammonium salts. Nutrient inorganic salts can also be incorporated in the medium so as to provide sodium, potassium, ammonium, calcium, phosphate, sulfate, nitrate, carbonate and like ions. Trace elements such as copper, manganese, iron zinc, etc. are added to the medium if desired, or they may be present as impurities of other constituents of the media. Submerged aerobic conditions are preferably employed for the production of large quantities of antibiotic, although for production of limited amounts, surface cultures and bottles may also be used. The general procedures used for the cultivation of other actinomycetes are applicable to the present invention.

Production of the antibiotics of the present invention can be effected by any temperature conducive to satisfactory growth of the producing organism, e.g. 18° to 39° C. and is conveniently carried out at a temperature of about 28° C. The fermentation may be carried out in flasks or in laboratory or industrial fermentors of various capacity.

When tank fermentation is to be used it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating a small volume of the culture medium with a slant, a cryopreservative culture or a lyophilized culture of the producing organism. After obtaining a viable and active inoculum in this manner, it is transferred aseptically to the fermentation tank charged with production medium for large scale production of the antibiotic of the present invention. The medium in which the vegetative inoculum is grown can be the same as, or different from, that utilized in the tank as long as it is such that a good growth of the producing organism is obtained and supplemented with the appropriate fluorotryptophan. Further agitation may be provided by a mechanical impeller. Antifoam agents such as lard oil or silicone oil may also be added if needed. Antibiotic production is monitored by high performance liquid chromatography assay or by conventional biological assay. In general, optimum production of the antibiotics of the present invention is achieved after incubation of about 6 days.

Isolation and purification of the so-obtained derivatives may be carried out by conventional chromatographic procedures.

Physical and Chemical Properties:

The compounds of Formulas IV, V and VIII have the following Physical and Chemical Properties:

Compound of Formula IV

Description: Bright yellow amorphous solid

Molecular Formula: C 26 H 19 F 2 N 3 O 7

Molecular weight: 523.454

Mass Spectrum: Kratos MS 25 Mass Spectrometer. FABMS 524 (M+H) + , 361 (M-162, loss of glucose).

Ultraviolet Spectrum: Hewlett Packard 845A Diode Array Spectrophotometer. Concentration 1.0 mg/100 ml MeOH. Neutral .sub.λ max nm (E 1%/1 cm): 405, 322(457), 288, 277, 259, 230(348).

Infrared Spectrum: Perkin-Elmer 1800 FTIR Spectrometer. KBr Pellet (cm -1 ): 3340, 2915, 1752, 1708, 1628, 1591, 1484, 1462, 1392, 1331, 1292, 1247, 1191, 1112, 1081, 1052, 947, 904 795, 762, 752, 752, 511.

360 MHz 1 H-NMR: Bruker Model AM-3000 Spectrometer. Duel carbon-proton probe, 5 mm. Solvent d 6 -DMSO. Observed chemical shifts (ppm): 11.76 (s,1H), 11.23(s,1H), 8.85(dd,1H), 8.77(dd,1H), 8.01(dd,1H), 7.68 (dd,1H), 7.46(m,2H), 6.29(d,1H), 6.12(t,1H), 5.45(d,1H), 5.17(d,1H), 4.95(d,1H), 4.07(d,1H), 3.95(m,2H), 3.81(d,1H), 3.59(m,2H).

90 MHz 13 C-NMR: Bruker Model AM-3000 Spectrometer. Proton decoupled spectrum. Duel carbon-proton probe, 5 mm. Solvent d 6 -DMSO. Observed chemical shifts (ppm): 170.9, 170.8, 157.0(d), 157.0(d), 138.6, 137.2, 130.7, 129.2, 121.7(d), 121.4(d), 121.3, 119.6, 116.6, 115.1, 115.0(d), 114.6(d), 113.3(d), 113.2(d), 109.1(d), 109.1(d), 84.8, 78.7, 76.5, 73.2, 67.6, 58.3.

Solubility: Soluble in DMSO, DMF, THF, acetone, EtOAc, MeOH.

Thin Layer Chromatography (R f values): Normal phase (silica gel 60); EtoAc: 0.10. EtoAc-MeOH (9:1 v/v): 0.41. Reversed phase (C 18 ); 0.1M NH 4 OAc-MeOH-CH 3 CN (2:4:4: v/v): 0.67

Compound of Formula V

Description: Bright yellow amorphous solid

Molecular Formula: C 26 H 19 F 2 N 3 O 7

Molecular Weight: 523.454

Mass Spectrum: Kratos MS 25 Mass Spectrometer. FABMS 524 (M+H) + , 361 (M-162, loss of glucose).

Ultraviolet Spectrum: Hewlett Packard 8452A Diode Array Spectrophotometer. Concentration 1.0 mg/100 ml MeOH. Neutral .sub.λ max nm (E 1%/1 cm): 398(60), 316(640), 280(259), 256(313), 226(526), (497).

Infrared Spectrum: Perkin-Elmer 1800 FTIR Spectrometer. KBr Pellet (cm -1 ): 3324, 2927, 1745, 1701, 1623, 1580, 1491, 1471, 1452, 1412, 1384, 1330, 1233, 1172, 1116, 1075, 1048, 1016, 963, 916, 829, 764, 745, 646, 635, 617, 498, 490.

360 MHz 1 H-NMR: Bruker Model AM-3000 Spectrometer. Duel carbon-proton probe, 5 mm. Solvent d 6 -DMSO. Observed chemical shifts (ppm): 11.77 (s,1H), 11.18(s,1H), 9.12(dd,1H), 9.05(dd,1H), 7.85(dd,1H), 7.43(dd,1H), 7.23(t,2H), 6.26(d,1H) , 6.24(s,1H), 5.31(d,1H), 5.04(d,1H), 3.98(m,2H), 3.87 (m,1H), 3.66(s,2H).

90 MHz 13 C-NMR: Bruker Model AM-3000 Spectrometer. Proton decoupled spectrum. Duel carbon-proton probe, 5 mm. Solvent d 6 -DMSO. Observed chemical shifts (ppm): 170.8, 170.7, 161.8(d), 161.8(d), 143.2(d), 141.5(d), 130.1, 128.7, 126.0(d), 125.9(d), 120.9, 119.3, 118.2, 118.1, 117.7, 116.5, 108.8(d), 108.6(d), 98.9(d), 98.3(d), 84.7, 78.6, 76.5, 73.1, 67.5, 58.3.

Solubility: Soluble in DMSO, DMF, THF, MeOH, acetone, EtOAc.

Thin Layer Chromatography (R f values): Normal phase (silica gel 60); EtoAc: 0.40. EtoAc-MeOH (9:1 v/v): 0.63. Reversed phase (C 18 ); 0.1M NH 4 OAc-MeOH-CH 3 CN (2:4:4: v/v): 0.60.

Compound of Formula VIII

Description: Bright yellow amorphous solid

Molecular Formula: C 27 H 21 F 2 N 3 O 7

Molecular Weight: 537.481

Mass Spectrum: Kratos MS 25 Mass Spectrometer. FABMS 538 (M+H) + , 361 (M-176, loss of 4-0-methylglucose).

Ultraviolet Spectrum: Hewlett Packard 8452A Diode Array Spectrophotometer. Concentration 1.2 mg/100 ml MeOH. Neutral .sub.λ max nm (E 1%/1 cm): 398(103), 316(1050), 280(387), 256(454), 228(780).

Infrared Spectrum: Perkin-Elmer 1800 FTIR Spectrometer. KBr Pellet (cm -1 ): 3324, 2938, 1747, 1703, 1623, 1580, 1491, 1471, 1452, 1412, 1384, 1330, 1233, 1172, 1140, 1116, 1087, 1054, 963, 918, 828, 764, 649, 635, 618, 498.

360 MHz 1 H-NMR: Bruker Model AM-3000 Spectrometer. Duel carbon-proton probe, 5 mm. Solvent d 6 -DMSO. Observed chemical shifts (ppm): 11.77 (s,1H), 11.18(s,1H), 9.12(dd,1H), 9.05(dd,1H), 7.88(dd,1H), 7.41(dd,1H), 7.23(m,2H), 6.25(d,1H), 6.24(s,1H), 5.36(dd,1H), 5.31(d,1H), 5.04(d,1H), 3.97(m,2H), 3.60-3.95(m,4H).

90 MHz 13 C-NMR: Bruker Model AM-3000 Spectrometer. Proton decoupled spectrum. Duel carbon-proton probe, 5 mm. Solvent d 6 -DMSO. Observed chemical shifts (ppm): 170.8, 170.7, 161.8(d), 161.7(d), 143.1(d), 141.5(d), 130.1, 128.7, 126.0(d), 125.9(d), 120.9, 119.4, 118.2, 118.1, 117.7, 116.5, 108.8(d), 108.6(d), 98.7(d), 98.3(d), 84.4, 77.2, 77.2, 76.2, 73.2, 59.9, 58.5 ppm.

Solubility: Soluble in DMSO, DMF, THF, acetone, EtOAc, MeOH.

Thin Layer Chromatography (R f values): Normal phase (silica gel 60); EtoAc: 0.72. EtoAc-MeOH (9:1 v/v): 0.89. Reversed phase (C 18 ); 0.1M NH 4 OAc-MeOH-CH 3 CN (2:4:4: v/v): 0.59.

Biological Properties:

Representative compounds of the present invention were tested against the transplanted mouse leukemia P388 to determine in vivo antitumor activity (Tables 1-3). CDF 1 mice were implanted intraperitioneally ("ip") with 10 6 P388 leukemia cells obtained from DBA/2 donor mice bearing this transplantable murine leukemia. The CDF 1 leukemic mice were treated ip with either saline (control mice) or doses of the compound of Formulas IV, V and VIII once at Day 1 post-tumor inoculation. These animals were observed daily and their deaths recorded. Average body weight changes (from the day of leukemia implant to the day of last treatment) were determined for all groups as a means of reflecting drug toxicity. The incidence of mice alive in each group on Day 5 post-tumor implant was recorded as an additional means of assessing drug toxicity. No therapeutic result was considered as meaningful if more than 1 mouse per treatment group had died by Day 5. Each treatment group consisted of 4 to 6 mice; control groups contained 10 mice. The number of mice, if any, surviving to Day 30 (the 1st day of the experiment) was also recorded.

Therapeutic efficacy was evaluated by determining the median survival time ("MST") of mice treated with the compound of Formula IV, V and VIII and comparing it to the MST of parallel control mice. This comparison was made by dividing the MST of the former by the latter and multiplying by 100 to derive a parameter called the percent T/C value. A percent T/C of ≧125% was considered to represent a meaningful increase in lifespan and, hence, an active result.

As shown in Table 1, the compound of Formula IV is active against P388 leukemia at dose levels ranging from 10 to 90 mg/kg. The best effect was achieved at a dosage of 30 mg/kg and consisted of a percent T/C of 175%. Toxicity was not observed even at the highest dose (90 mg/kg) tested.

As shown in Table 2, the compound of Formula V is active against P388 leukemia at dose levels ranging from 0.8 to 102.4 mg/kg. The best effect was achieved at a dosage of 102.4 mg/kg and consisted of a percent T/C of 178%. Toxicity was not observed even at the highest dose (102.4 mg/kg) tested.

As shown in Table 3, the compound of Formula VIII is active against P388 leukemia at dose levels ranging from 0.8 to 102.4 mg/kg. The best effect was achieved at a dosage of 51.2 mg/kg and consisted of a percent T/C of 206%. Toxicity was not observed at the highest dose (102.4 mg/kg) tested.

______________________________________

Effect of Compound of Formula IV on P388 Leukemia.sup.a

(Day 1 Treatment)

Median Average No. of Mice

Dose, ip.

Survival % Weight Alive on

(mg/kg/inj)

Times (Days)

T/C Change (g)

Day 5 Day 30

______________________________________

90 16.5 165 -0.9 4/4 0/4

30 17.5 175 0.6 4/4 0/4

10 14.5 145 0.7 4/4 0/4

Control 10.0 100 10/10 0/10

______________________________________

.sup.a Mice were implanted with 10.sup.6 P388 leukemia cells, and

treatments with the compound of Formula IV were begun 1 day later. Contro

mice were given saline injections.

______________________________________

Effect of Compound of Formula V on P388 Leukemia.sup.a

(Day 1 Treatment)

Median Average No. of Mice

Dose, ip.

Survival % Weight Alive on

(mg/kg/inj)

Times (Days)

T/C Change (g)

Day 5 Day 30

______________________________________

102.4 16.0 178 -0.1 6/6 0/6

51.2 15.0 167 -0.2 6/6 0/6

25.6 14.5 161 -0.2 6/6 0/6

12.8 15.0 167 0.8 6/6 0/6

6.4 15.0 167 -1.1 6/6 0/6

3.2 14.0 156 0.0 6/6 0/6

1.6 14.0 156 -0.7 6/6 0/6

0.8 13.0 144 0.1 6/6 0/6

Control 9.0 100 1.3 10/10 0/10

______________________________________

.sup.a Mice were implanted with 10.sup.6 P388 leukemia cells and

treatments with the compound of Formula V were begun 1 day later. Control

mice were given saline injections.

______________________________________

Effect of Compound of Formula VIII on P388 Leukemia.sup.a

(Day 1 Treatment)

Median Average No. of Mice

Dose, ip.

Survival % Weight Alive on

(mg/kg/inj)

Times (Days)

T/C Change (g)

Day 5 Day 30

______________________________________

102.4 14.5 161 0.2 6/6 0/6

51.2 18.5 206 0.3 6/6 0/6

25.6 14.0 156 0.1 6/6 0/6

12.8 14.5 161 -0.1 6/6 0/6

6.4 16.0 178 0.1 6/6 0/6

3.2 16.5 183 -0.3 6/6 0/6

1.6 14.0 156 -0.1 6/6 0/6

0.8 14.0 156 -0.4 6/6 0/6

Control 9.0 100 1.3 10/10 0/10

______________________________________

.sup.a Mice were implanted with 10.sup.6 P388 leukemia cells and

treatments with the compound of Formula VIII were begun 1 day later.

Control mice were given saline injections.

The present invention includes within its scope pharmaceutical compositions which comprise an effective or tumor-inhibiting amount of a rebeccamycin analog of the present invention, or a pharmaceutically acceptable acid addition salt thereof, in combination with an inert pharmaceutically acceptable carrier or diluent.

According to another aspect of the invention, a method is provided for therapeutically treating an animal (preferably mammalian) host effected by a malignant tumor which comprises administering to such host an effective tumor-inhibiting dose of the antibiotic of the present invention or a pharmaceutically acceptable acid addition salt thereof.

Examples of suitable compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups and elixirs and preparations for parenteral administration such as sterile solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline or some other sterile injectable medium immediately before use.

It will be appreciated that the actual preferred dosages of the rebeccamycin analogs of the present invention will vary according to the particular compound being used, the particular composition formulated, the mode of application and the particular situs, host and disease being treated. Many factors that modify the action of the drug will be taken into account by those skilled in the art, e.g. age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease. Administration can be carried out continuously or periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be readily ascertained by those skilled in the art using conventional dosage determination tests.

The present invention is illustrated by the following examples which are not intended to be construed as limiting the scope of the invention.